Methods and compositions for preparing polynucleotide libraries

ABSTRACT

Provided herein are methods of producing molar amounts of nucleic acids in a sample by using fixed molar amounts of primers and amplifying nucleic acids in the sample through a sufficient number of rounds to use all the primer. The methods may be used on a plurality of separate samples to produce samples with molar amounts of nucleic acids that are the same, or substantially the same, or that are related in known molar ratios. Further provided herein are normalized, optionally pooled nucleic acid libraries.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional PatentApplication No. 62/586,649, entitled “Methods Of Preparing Nucleic AcidLibraries”, filed Nov. 15, 2017, and U.S. Provisional Patent ApplicationNo. 62/652,613, entitled “Methods For Preparing PolynucleotideLibraries”, filed Apr. 4, 2018, both of which are incorporated herein byreference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

None.

BACKGROUND

In various applications, it can be useful to control and/or quantify theamount, e.g., the molar amount, of nucleic acid in a sample. In highthroughput sequencing methods, nucleic acid libraries produced from eachof a plurality of different samples may be pooled and sequencedtogether, for the sake of economy. Typically, in such situations, thelibraries are indexed with barcodes so that polynucleotides fromdifferent libraries can be distinguished upon sequencing.

It may be useful to achieve molar concentrations of nucleic acids indifferent libraries that are equimolar or that are related as a knownratio of molarities. Ultimately, a pool comprising different librariesis produced, each library in the pool having a molar amount of nucleicacids that is equal to other libraries, or related to other libraries ina known molar ratio

SUMMARY

In one aspect, provided herein are methods and compositions forachieving molar concentrations of nucleic acids in different librariesthat are equimolar or that are related as a known ratio of molarities.

Provided herein are methods of amplifying nucleic acids comprising a)performing a plurality of primer extension reactions on nucleic acidmolecules in each of a plurality of separate nucleic acid samplescontaining nucleic acids, using first polynucleotide primers to initiateprimer extension in each sample, wherein: 1) each of the plurality ofsamples is provided with a fixed molar amount of first primer; 2) thefixed molar amount of first primer in each sample is the same orsubstantially the same, or is related to the molar amount in othersamples in a known ratio; and 3) the plurality of primer extensionreactions in each sample is such that, upon completion of the pluralityof primer extension reactions, all first primer in each sample has beenused to produce amplified nucleic acids. The molar amount of firstprimer in the samples may be the same or substantially the same. Themethod can further comprise using a second polynucleotide primer,wherein the second primer is present in equal or substantially equalmolar amount as the first primer, or in a known greater molar amount asthe first primer. The second primer may be present in equal orsubstantially equal molar amount as the first primer, and after theplurality of primer extension reactions the molar amount of amplifiednucleic acid may be equal to or substantially equal to the molar amountof first and/or second primer. The first and second primers can bedifferent. The first and the second primers can be the same. In certainembodiments, the method further comprises b) combining a portion of eachof the plurality of separate samples to provide a pooled samplecomprising a library of amplified nucleic acids, wherein the molaramount of amplified nucleic acid from each separate sample in the pooledsample is the same or substantially the same, or related to the molaramounts of other nucleic acids in the pooled sample in a known ratio,thus producing a pooled nucleic acid library. In some cases, the portionof at least one of the plurality of samples is all of the sample. Insome cases, the portion of at least two of the plurality of samples isthe same. In some cases, the portions of at least two of the pluralityof samples is different. The plurality of primer extension reactions cancomprise a plurality of rounds of polymerase chain reaction (PCR)amplification, loop-mediated isothermal amplification (LAMP), reversetranscription loop-mediated isothermal amplification (RT-LAMP),strand-displacement amplification (SDA), helicase-dependentamplification (HDA), or transcription-mediated amplification (TMA). Incertain embodiments, the nucleic acid molecules in the plurality ofseparate samples comprise DNA molecules, for example cDNA molecules. Incertain embodiments, the nucleic acid molecules in the plurality ofseparate samples comprise RNA molecules. In certain embodiments, theamplified nucleic acids comprise a sample barcode comprising apredetermined nucleotide sequence, wherein the barcode is different foreach different nucleic acid sample. In certain embodiments, theplurality of samples comprises at least 2, at least 5, at least 10, atleast 20, at least 50, or at least 100 separate samples.

Also provided herein are methods for amplifying nucleic acidscomprising: a) providing a plurality of samples comprising RNA; b)performing a first round of cDNA synthesis on the RNA in the samples toproduce a library of first strand cDNA molecules; c) performing a secondround of cDNA synthesis on the cDNA strand produced in the first roundto provide second strand cDNA molecules, wherein the second strand cDNAmolecules comprise a first primer binding site for a first primer and atemplate for a second primer binding site for a second primer ; and d)amplifying the cDNA produced in step c) by providing each of theplurality of samples a molar amount of first primer and second primer,wherein the molar amount of the second primer is equal to orsubstantially equal to the molar amount of the first primer, or isgreater than the molar amount of the first primer in a known amount, andperforming a plurality of primer extension reactions, wherein 1) themolar amount of first primer in each of the plurality of samples is thesame or substantially the same, or is related to the molar amount inother samples in a known ratio; and 2) wherein the molar amount of firstprimer in each sample is such that, upon completion of the plurality ofprimer extension reactions, all first primer in each sample has beenused. The first primer can comprise a first polynucleotide sequence andthe second primer can comprise a second polynucleotide sequence, whereinthe first and second polynucleotide sequences are the same, and whereinthe molar amount of first and second primer in each sample is the same.At least one of the primers can comprise sequencing platform-specificadapter sequences; the sequencing platform specific sequences compriseone or more of a sequencing primer hybridization site, a sample barcodeand a cluster primer binding site. The method can further comprise e)preparing a pooled nucleic acid library by combining a portion of eachof the plurality of samples comprising amplified nucleic acid. In somecases, the portion of at least one of the samples comprises all of thesample. The method can further comprise f) sequencing the amplifiednucleic acids in the pooled library. The primer extension reactions cancomprise a plurality of rounds of PCR, LAMP, RT-LAMP, SDA, HDA, or TMA.In some cases, the predetermined molar amount of first primer in each ofthe plurality of samples is the same or substantially the same.

Also provided herein are methods comprising a) providing a plurality ofseparate samples wherein each sample comprises adapter-taggedpolynucleotides, wherein the adapter-tagged polynucleotides comprise apolynucleotide insert flanked by adapter sequences comprising firstprimer binding sites; b) amplifying the adapter-tagged polynucleotidesin each of the plurality of samples using primers in each sample thatbind to the first primer binding sites and performing a plurality ofprimer extension reactions to produce amplified polynucleotides, whereinthe first primer added to each sample is added in a predetermined molaramount, wherein 1) the molar amount of first primer in each sample isthe same or substantially the same, or is related to the molar amount inother samples in a known ratio; and 2) the molar amount of first primerin each sample is such that, upon completion of the plurality of primerextension reactions, all first primer in each sample has been used. Themethod can further comprise c) combining a portion of each of theplurality of separate samples to provide a pooled sample comprising alibrary of amplified nucleic acids, wherein the molar amount of eachdifferent amplified nucleic acid in the library is the same orsubstantially the same, or related to the molar amounts of otheramplified nucleic acids in the library in a known ratio, thus producinga pooled nucleic acid library. The portion of at least one of thesamples can comprise all of the sample. In certain embodiments,providing adapter-tagged polynucleotides comprises performing primerextension of polynucleotides using primers comprising adapter sequencesor ligating adapters to double stranded polynucleotides. In some cases,the adapter-tagged polynucleotides further comprise a second primerbinding site for a second primer, and wherein the second primer is addedin equal or substantially equal amount as the first primer, or in knowngreater molar amount as the first primer. The first and second primerbinder sites can comprise the same binding sequence and the first andsecond primers can bind to the binding sequence, and the first andsecond primers can be present in the same or substantially the samemolar amount. In some cases, the adapter sequences comprise samplebarcodes. In some cases, the polynucleotides comprise adapter sequencescomprising sequencing platform-specific sequences necessary and/orsufficient for sequencing the amplified polynucleotides; the sequencingplatform-specific sequences can comprise one or more of a sequencingprimer hybridization site, a sample barcode and a cluster primer bindingsite. In some cases the method further comprises d) sequencing amplifiednucleic acids in the nucleic acid library.

Further provided herein are methods comprising a) preparing a set ofnucleic acid libraries by amplifying nucleic acids in a plurality ofseparate samples comprising nucleic acids so that the amplified nucleicacids are present in each of the plurality of separate samples in thesame or substantially the same molar amount, or are present in molaramounts in known ratios to each other; and b) combining a portion ofeach of the separate samples to produce a pool of nucleic acid librariesin which the molar amounts of amplified nucleic acids from each nucleicacid library in the pool are the same or substantially the same, or arepresent in molar amounts in known ratios to each other. The portion ofat least one of the samples can comprise all of the sample. The methodcan further comprise c) sequencing the nucleic acids in the pool ofnucleic acid libraries. In some cases, the amplified nucleic acids inthe separate samples comprise a barcode, wherein the barcode for anynucleic acid library is the same for nucleic acids in that library anddifferent from the barcodes for other nucleic acid libraries.

Further provided herein are methods of producing a nucleic acid librarycomprising a) providing a plurality of samples comprising nucleic acids;b) adding adapter sequences to the nucleic acids in each sample, whereinthe adapter sequences comprise primer binding sites, and wherein thenucleotide sequence of the primer binding sites of the adapters aredifferent for each sample; c) combining a portion or all of the samplesto produce a pooled sample; d) performing a plurality of primerextension reactions on nucleic acid molecules in the pooled sample,using primers specific to the primer binding sites of the adapters,wherein the different primers are present in equal or substantiallyequal molar amounts, or in known ratios of molar amounts, wherein theplurality of primer extension reactions is sufficient to use all orsubstantially all of the primers to produce amplified nucleic acids;thereby producing a final sample in which the amplified nucleic acidsfrom the different original samples are present in the same orsubstantially the same molar amounts, or in molar amounts of knownratio. The adapter sequences can further comprise a barcode, wherein thebarcode is different for each sample. The adapter sequences can furthercomprise sequencing platform-specific adapter sequences

Also provided herein are compositions.

In certain embodiments, provided herein is a set of a plurality ofseparate nucleic acid libraries, wherein: a) each library comprisespolynucleotides; and b) molar amounts of polynucleotide molecules fromeach nucleic acid library in the set are the same or substantially thesame, or are related as a known ratio to each other. In certainembodiments, each separate nucleic acid library is in a separatecontainer. The set may also be distinguished in that b) thepolynucleotides comprise a sample barcode, and polynucleotides in anylibrary have the same sample barcode; and c) sample barcodes indifferent libraries are different. In certain embodiments, the molaramounts of polynucleotide molecules in each library are the same orsubstantially the same. In certain embodiments the polynucleotidescomprise RNA. In certain embodiments the polynucleotides comprise DNA.

In certain embodiments, provided herein is a kit comprising a pluralityof sets of polynucleotide primers, wherein each set is contained in aseparate container or containers from the other sets, and wherein eachset comprises first polynucleotide primers and second polynucleotideprimers, wherein: a) the molar amount of the first polynucleotideprimers in each set is equal or substantially equal, or is in knownratio to the molar amount of first polynucleotide primers in other sets;and b) the molar amount of the second primer in each set is equal to orsubstantially equal to the molar amount of the first primer in that set,or greater than the molar amount of the first primer in that set by aknown amount. The first primer and the second primer can have differentpolynucleotide sequences. The first primer and the second primer canhave the same polynucleotide sequences and the first and second primerscan be present in equimolar concentrations. The kit can further comprisereagents for performing nucleic acid amplification, such as reagents forPCR, LAMP, RT-LAMP, SDA, HDA, or TMA. In some cases, the firstpolynucleotide primers and the second polynucleotide primers for eachset of primers are contained in the same container. In some cases, thefirst polynucleotide primers and the second polynucleotide primers foreach set of primers are contained in different containers.

In certain embodiments, provided herein is a kit comprising a) aplurality of sets of polynucleotide adapters comprising primer bindingsites, wherein each set is contained in a separate container from theother sets, and b) one or more primers for binding to the primer bindingsites for nucleic acid amplification. The primer binding sites of eachset of polynucleotide adapters can the same. The primer or primers canbe contained in a single container. The kit can further comprisereagents for performing nucleic acid amplification, such as reagents forPCR, LAMP, RT-LAMP, SDA, HDA, or TMA.

In another aspect, provided herein are methods and compositions fornormalizing nucleic acids in different libraries by using detectablelabels.

Provided herein is a method comprising: a) performing one or a pluralityof primer extension reactions on nucleic acid molecules in a sampleusing polynucleotide primers, at least a portion of which polynucleotideprimers bear a detectable label, to produce a nucleic acid librarycomprising synthesized nucleic acids, at least a portion of whichsynthesized nucleic acids incorporate the detectable label; and b)measuring an amount of detectable label incorporated into synthesizednucleic acids in the library, wherein amount of incorporated labelindicates an amount of synthesized nucleic acids in the library. In oneembodiment no more than one primer extension reaction usespolynucleotide primers bearing a detectable label. In another embodimentthe primer extension reaction using labeled polynucleotide primersperforms first strand synthesis of a first cDNA molecule from an RNAmolecule. In another embodiment all or substantially all of thepolynucleotide primers bear the detectable label. In another embodimentno more than 75% of the polynucleotide primers bear the detectablelabel. In another embodiment a plurality of the primer extensionreactions use polynucleotide primers bearing the detectable label. Inanother embodiment the plurality of primer extension reactions comprisea plurality of rounds of PCR amplification. In another embodiment themethod further comprises before determining, separating unincorporatedlabeled primers from the synthesized nucleic acids. In anotherembodiment the detectable label is an optical label. In anotherembodiment the detectable label is a fluorescent label. In anotherembodiment the fluorescent label is phycoerythrin. In another embodimentthe detectable label is a fluorescent label and quantifying comprisesinducing fluorescence from the fluorescent label and measuringfluorescence. The synthesized nucleic acids comprise cDNA sequences. Inanother embodiment the synthesized nucleic acids comprise a samplebarcode comprising a predetermined nucleotide sequence. In anotherembodiment the method further comprises cleaving the incorporateddetectable label from the synthesized nucleic acids. In anotherembodiment primers are provided in pairs comprising forward primers andreverse primers, and only one member of a pair comprises the detectablelabel. In another embodiment more than 50% of the primers comprise thedetectable label. In another embodiment no more than 50%, no more than25%, no more than 10% or no more than 5% of the primers comprise thedetectable label. In another embodiment the method further comprises: c)preparing a sample comprising the nucleic acid library, wherein thenucleic acid library comprises a predetermined amount of the synthesizednucleic acids, e.g., an amount normalized with respect to at least oneother nucleic acid library. In another embodiment the synthesizednucleic acid molecules in the nucleic acid library comprise at least onesample barcode. In another embodiment the detectable label is removedfrom the synthesized nucleic acids before or after normalizing. Inanother embodiment the method is performed on a plurality of differentsamples comprising nucleic acid to produce a plurality of nucleic acidlibraries, and the method further comprises: c) preparing a pooled,normalized nucleic acid library comprising normalized amounts ofsynthesized nucleic acids from the plurality of nucleic acid libraries.In another embodiment nucleic acid molecules in each nucleic acidlibrary comprise a sample barcode and wherein the sample barcodes aredifferent between different samples. In another embodiment thedetectable label is removed from the synthesized nucleic acids before orafter preparing the pooled, normalized nucleic acid library. In anotherembodiment the plurality of samples is at least 10, at least 20, atleast 50, or at least 100. In another embodiment preparing the pooled,normalized nucleic acid library comprises preparing a plurality ofnormalized nucleic acid libraries and pooling same volumes of thenormalized libraries. In another embodiment preparing the pooled,normalized nucleic acid library comprises pooling different volumes of aplurality of libraries, wherein each volume comprises equimolar amountsof nucleic acid.

Also provided herein is a method comprising: a) providing a samplecomprising RNA; b) performing first strand cDNA synthesis on the RNAusing first primers to produce a library of first strand cDNA molecules,wherein the primers comprise sequencing platform-specific adaptersequences and at least a portion of the primers comprise a detectablelabel; c) quantifying an amount of first strand cDNA molecules in thelibrary by measuring an amount of the detectable label incorporated inthe cDNA, wherein the amount of cDNA is a function of the amount ofincorporated label. In one embodiment the sequencing platform specificsequences comprise one or more of a sequencing primer hybridizationsite, a sample barcode and a cluster primer binding site. In anotherembodiment performing first strand synthesis further comprises, afterprimer extension, extending the first strand using a template moleculethat hybridizes to the first strand and that comprises sequencingplatform-specific adapter sequences. In another embodiment the methodfurther comprises: d) preparing a normalized nucleic acid librarycomprising an amount of first strand cDNA molecules normalized topredetermined amount. In another embodiment the method furthercomprises: d) sequencing the first strand cDNA molecules, e.g., withoutfurther amplification.

Further provided herein is a method comprising: a) quantifying,according to any method provided herein, amounts of second strand cDNAmolecules in each of a plurality of nucleic acid libraries; and b)preparing a pool of normalized nucleic acid libraries in which theamounts of second strand cDNA molecules from each nucleic acid libraryin the pool are normalized with respect to one another. In oneembodiment the method further comprises: c) sequencing the pool ofnormalized nucleic acid libraries. In another embodiment sequencing isperformed without amplification of the normalized nucleic acid librariesin the pool. In another embodiment the normalized nucleic acid librariesin the pool are amplified before sequencing.

Further provided herein is a method for comprising: a) providing asample comprising adapter-tagged polynucleotides, wherein theadapter-tagged polynucleotides comprise a polynucleotide insert flankedby adapter sequences comprising first primer binding sites; b)amplifying the adapter-tagged polynucleotides using primers to produce anucleic acid library of amplified polynucleotides, wherein at least aportion of the primers comprise a detectable label and wherein at leasta portion of the amplified polynucleotides incorporate the detectablelabel; and c) quantifying an amount of amplified polynucleotides in thenucleic acid library by measuring an amount of the detectable labelincorporated in the amplified polynucleotides, wherein the amount ofamplified polynucleotides is a function of the amount of incorporatedlabel. In one embodiment providing adapter-tagged polynucleotidescomprises performing primer extension of polynucleotides using primerscomprising adapter sequences or ligating adapters to double strandedpolynucleotides. In another embodiment the first primer binding sitescomprise a first forward primer binding site and a first reverse primerbinding site, wherein the first forward primer binding site and thefirst reverse primer binding site are the same or different. In anotherembodiment the primers comprise a first primer set and a second primerset, wherein: (i) primers in the first primer set comprise sequencesthat bind to the first primer binding sites and further comprise secondprimer binding sites comprising a second forward primer binding site anda second reverse primer binding site, wherein the second forward primerbinding site and the second reverse primer binding site are the same ordifferent; and (ii) primers in the second primer set comprise sequencesthat bind to the second primer binding sites, and wherein at least aportion of the primers in the second primer set comprise the detectablelabel. In another embodiment the first set and second set of primerpairs are present in amounts having relative ratios between about 2:1 toabout 1:2, e.g., about 1:1. In another embodiment: (i) the first forwardand reverse primer binding sites are different; and (ii) the secondforward and reverse primer binding sites are different. In anotherembodiment the first set of primers comprise sample barcodes. In anotherembodiment the amplified polynucleotides comprise adapter sequencescomprising sequencing platform-specific sequences necessary and/orsufficient for sequencing the amplified polynucleotides. In anotherembodiment the sequencing platform specific sequences comprise one ormore of a sequencing primer hybridization site, a sample barcode and acluster primer binding site. In another embodiment the method furthercomprises: d) preparing a normalized nucleic acid library comprising anamount of amplified polynucleotides normalized to a predeterminedamount. In another embodiment the method further comprises: e)sequencing amplified polynucleotides in the nucleic acid library.

Further provided herein is a method comprising: a) quantifying,according to any method as provided herein, amounts of amplifiedpolynucleotides in each of a plurality of nucleic acid libraries; and b)preparing a pool of normalized nucleic acid libraries in which theamounts of amplified polynucleotides from each nucleic acid library inthe pool are normalized with respect to one another. In anotherembodiment the method further comprises: c) sequencing the pool ofnormalized nucleic acid libraries.

Further provided herein is a pool of normalized nucleic acid libraries,wherein: a) each library comprises polynucleotides, at least a portionof which polynucleotides in each library comprise a detectable label; b)polynucleotides in any library have the same sample barcode; c) samplebarcodes in different libraries are different; d) amounts ofpolynucleotide molecules from each nucleic acid library in the pool arenormalized with respect to one another.

Further provided herein is a collection of nucleic acid libraries, eachlibrary contained in a different container, wherein at least a portionof polynucleotides in each library comprises a detectable label. Inanother embodiment polynucleotides in each nucleic acid library comprisea sample barcode that distinguishes polynucleotides in one nucleic acidlibrary from those in another nucleic acid library. In anotherembodiment amounts of polynucleotides in each library are normalizedwith respect to each other.

Further provided herein is a kit comprising: a) a first primer setcomprising primers comprising sequences that bind to first primerbinding sites and comprising a detectable label; and (b) a second primerset comprising primers having the same nucleotide sequence as primers inthe first set, and not comprising a detectable label; wherein the ratioof primers in the first set to primers in the second set is betweenabout 1:100 to 100:1, between about 10:1 to 1:10, between about 1:5 to5:1, between about 1:3 to 3:1, between about 1:2 to 2:1 or about 1:1, orwherein there are fewer primers in the first primer set than in thesecond primer set. In one embodiment the kit further comprises atemplate polynucleotide comprising a 3′ terminal nucleotide sequencethat binds to an overhang produced by a polymerase, wherein the templatepolynucleotide comprises a primer binding site. In another embodimentthe kit further comprises a reverse transcriptase. In another embodimentthe kit further comprises reagents for performing primer extension,including nucleotides and buffers. In another embodiment the kit furthercomprises one or more containers that contain the first primer set andthe second primer set in the same or separate containers.

Further provided herein is a kit comprising first polynucleotideadapters and second polynucleotide adapters, wherein: (a) the firstpolynucleotide adapters comprise (i) first binding sequences for bindingto a target primer binding site and (ii) primer binding sites forbinding to binding sequences on the second polynucleotide adapters,wherein the first polynucleotide adapters do not comprise a detectablelabel; and (b) the second polynucleotide adapters comprise secondbinding sequences for binding to the primer binding sites on the firstpolynucleotide adapters, wherein at least a portion of the secondpolynucleotide adapters comprise a detectable label. In one embodimentthe first polynucleotide adapters comprise forward primers and reverseprimers, wherein the forward primers comprise forward binding sequencesand forward primer binding sites and the reverse primers comprisereverse binding sequences and forward primer binding sites; and thesecond polynucleotide adapters comprise forward primers and reverseprimers, wherein the forward primers comprise forward primer bindingsequences and the reverse primers comprise reverse binding sequences. Inanother embodiment the kit further comprises reagents for performingPCR, including a DNA polymerase, nucleotides and buffers. In anotherembodiment the first primer set and the second primer set are containedin the same or separate containers.

Other objects of the disclosure may be apparent to one skilled in theart upon reading the following specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 shows an exemplary protocol for preparing nucleic acids forprimer extension by the methods disclosed herein.

FIG. 2 shows an exemplary method of producing a first strandadapter-tagged cDNA molecule for primer-limited amplification.

FIG. 3 shows an exemplary method of producing an adapter-tagged cDNAmolecule from an RNA molecule. The adapters comprise primer bindingsites (PBS1 and PBS2′) which further function as amplification bindingsites.

FIG. 4 shows an exemplary method of amplifying the adapter-taggednucleic acid using two sets of forward and reverse primers, in aprimer-limited amplification. A (phantom) complement of a cDNA strand isshown, for ease of understanding, as a dotted line. Not shown areproducts of first forward extension and first reversed extension.

FIG. 5 shows an exemplary protocol for generating sequence reads from apool of normalized nucleic acid libraries produced by primer-limitedamplification.

FIG. 6 shows an exemplary method of producing a first strandadapter-tagged cDNA molecule comprising a detectable label.

FIG. 7 shows an exemplary method of amplifying the adapter-taggednucleic acid using two sets of forward and reverse primers, in aamplification with normalization by labeling. A (phantom) complement ofa cDNA strand is shown, for ease of understanding, as a dotted line. Notshown are products of first forward extension and first reversedextension.

FIG. 8 shows an exemplary protocol for generating sequence reads from apool of normalized nucleic acid libraries produced by labeling.

FIG. 9 shows the number of reads as a percentage of the mean for 1125samples amplified according to the primer-limited methods describedherein then read on an Illumina sequencer; see Example 3.

DETAILED DESCRIPTION I. Definitions

As used herein, the term “sample” includes a composition comprising ananalyte. A sample can be a raw sample, in which the analyte is mixedwith other materials in its native form (e.g., a source material), afractionated sample, in which an analyte is at least partially enriched,or a purified sample in which the analyte is at least substantiallypure.

As used herein, a chemical entity, such as a polynucleotide orpolypeptide, is “substantially pure” if it is the predominant chemicalentity of its kind in a composition. This includes the chemical entityrepresenting more than 50%, more than 80%, more than 90% or more than95% or of the chemical entities of its kind in the composition. Achemical entity is “essentially pure” if it represents more than 98%,more than 99%, more than 99.5%, more than 99.9%, or more than 99.99% ofthe chemical entities of its kind in the composition. Chemical entitieswhich are essentially pure are also substantially pure.

As used herein, the term “subject” includes an individual organism,e.g., an animal, a plant or a microbe. Animal subjects include, withoutlimitation, human and nonhuman animals. Nonhuman animals may be mammals,birds, fish, reptiles and insects. Nonhuman animals include, forexample, bovines, swine, horses, sheep, goats, chickens, turkeys, dogs,cats and birds.

As used herein, the term “host” includes an organism hosting a microbialcommunity.

As used herein, the term “microbiome” includes a microbial communitycomprising one or a plurality of different microbial strains or speciesinhabiting a host.

As used herein, the terms “polynucleotide” and “nucleic acid” are usedinterchangeably and include both single-stranded and double-strandedmolecules. As used herein, the term “oligonucleotide” includes shortpolynucleotides, e.g., no more than 500 nucleotides in length. Incertain embodiments, a polynucleotide can comprise natural ornon-natural nucleotides, such as peptide nucleic acids or locked nucleicacids.

As used herein, “cell-free nucleic acid” (e.g., “cell-free DNA”(“cfDNA”) or “cell-free RNA”) includes nucleic acid not encapsulated ina cell and found in a bodily fluid, such as blood/serum/plasma, urine,amniotic fluid, saliva, pleural effusion, bronchial lavage or aspirates,breast milk, colostrum, tears, seminal fluid, peritoneal fluid, andstools. Typically, cell-free DNA comprises DNA having a size rangebetween about 140 and about 180 nucleotides.

As used herein, the term “adapter” includes a polynucleotide comprisingadapter sequences comprising, at least, a primer binding site, e.g., auniversal primer binding site or a forward or reverse primer bindingsite. Adapters also can comprise other elements including, withoutlimitation, a sample barcode, a molecular barcode, a sequencing primerbinding site (which may also serve as an amplification primer bindingsite) or a binding site for binding polynucleotide to platform hardware,such as a flow cell probe binding site. In certain embodiments, adapterscan comprise non-complementary ends. These include, for example,“Y-shaped” adapters or adapters which fold back upon themselves to formlooped structures. Y-shaped adapters, in particular, can be useful whendifferent strands (“Watson” and “Crick” strands) of a double strandednucleic acid need to be distinguished. Depending on context, the term“adapter” may also refer to a nucleotide sequence comprising adapterelements.

As used herein, the term “adapter-tagged polynucleotide” includes apolynucleotide comprising a nucleic acid insert flanked on one or bothends by adapter sequences bearing a primer binding site.

As used herein, the term “nucleic acid library” includes a collection ofnucleic acids, for example, a collection of adapter-taggedpolynucleotides. Typically, polynucleotide members of a nucleic acidlibrary comprise a sample index. Optionally, they may comprise molecularbarcodes useful for distinguishing individual molecules from each other,either using the barcode, alone, or in combination with insert sequenceinformation.

As used herein, the term “primer binding site” includes a nucleotidesequence to which a polynucleotide primer can hybridize, e.g., for PCRor primer extension.

As used herein, the term “primer” includes a polynucleotide, typicallyan oligonucleotide, having a sequence (“binding sequence”) that binds toa primer binding site. Primers are typically categorized as “universalprimers” or “degenerate primers”. Primers are used for primer extensionand PCR. In amplification, such as PCR, primers bind to primer bindingsites on each strand of a double stranded nucleic acid molecule with atarget sequence (amplicon) positioned between them. In certainembodiments, for example, when the primer binding site on the firststrand of a double stranded molecule is different than the primerbinding site on a second, complementary, strand, primers are provided asa set of two primers (“primer pair”). Primers in the primer pair may bedifferentiated as a “forward primer” and a “reverse primer”.

As used herein, the term “universal primer” includes a primer having abinding sequence that binds to a primer binding site on an adapter.Accordingly, a universal primer can be used to amplify alladapter-tagged polynucleotides in a sample.

As used herein, the term “degenerate primer” includes a mixture ofprimers having a substitution of different nucleotides at the bindingsequence. For example, degenerate primers can have a degenerate hexamernucleotide sequence.

As used herein, the term “barcode” includes a nucleotide sequence whichprovides information about the polynucleotide in which the barcode isincorporated. A barcode may provide information specific to a singlemolecule or collection of molecules. Barcodes are typically provided inpolynucleotide adapters. Barcodes typically have sequences of no morethan 100, 50, 20 or 10 nucleotides.

As used herein, the term “sample barcode” includes a barcode thatdistinguishes polynucleotides sourced from a first sample frompolynucleotides sourced from a second, different sample. Accordingly,sample barcodes in an ensemble of adapters will be the same in eachsample and different between different samples. For example,polynucleotides sourced from each of 50 different samples may comprise50 different sample barcodes.

As used herein, the term “molecular barcode” includes a barcode that,alone or in combination with other information, distinguishes differentmolecules in a sample from each other. For example, a set of molecularbarcodes may have sufficient diversity such that substantially allmolecules in a sample bear a different molecular barcode. A collectionof such polynucleotides is referred to as being “uniquely tagged”.Alternatively, a set of barcodes may have a diversity that is less thanthe number of polynucleotides in a sample. In this case, differentmolecules that bear the same molecular tag may be distinguished based oninformation derived from the sequence of the insert. A collection ofsuch polynucleotides is referred to herein as being “non-uniquelytagged”.

As used herein, the term “index” includes one or more pieces ofinformation, such as barcodes, which, alone or in combination, provideinformation. For example, an adapter-tagged polynucleotide can comprisea single sample barcode and/or molecular barcode, or a plurality ofsample barcodes or molecular barcodes, e.g., attached at each end. Asingle barcode or a collection of barcodes attached to a molecule canfunction as an “index”. Thus, a “sample index” can be defined by one ora plurality (e.g., two) of sample barcodes, and a “molecular index” canbe defined by one or a plurality (e.g., two) of molecular barcodes.

As used herein, the term “detectable label” includes a compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,chemical, or other physical means. Examples of detectable labelsinclude, without limitation, colorimetric, fluorescent,chemiluminescent, enzymatic, and radioactive labels. A detectable labelcan produce a signal directly (a “direct label”) or indirectly (an“indirect label”). A direct label directly produces a signal. Examplesof direct labels are fluorescent labels (e.g., phycoerythrin,fluorescein isothiocyanate, texas red, rhodamine, a green fluorescentprotein, a red fluorescent protein, a yellow fluorescent protein),luminescent labels (e.g., luminescent proteins such as luciferase),enzymatic labels (e.g., horse radish peroxidase or alkalinephosphatase), colorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beadsand radioactive labels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P). In oneembodiment, the detectable label is a molecular beacon comprising anucleotide hairpin structure having tethered to its ends a fluorophoreand a quencher. An indirect label is a label that is detected (primarilyor secondarily) by another moiety comprising a direct label. Examples ofindirect labels are capture moieties, such as antibodies, biotin orstreptavidin, that bind other molecules which themselves bear a directlabel.

Detectable labels can be measured as follows. Fluorescence: Afluorescent molecule (fluorophore), such as a dye or a protein, areexcited with light of specific wavelength. The fluorophore then emitslight of a specific wavelength, which can be measured using a detector,such as a photomultiplier tube, CMOS, etc. Luminescence: Chemicalreactions can produce light. One example is enzyme Luciferase thatoxidizes luciferin and emits photons. This light can be measured using adetector, such as a photomultiplier tube, CMOS, etc.

As used herein, the term “high throughput sequencing” includes thesimultaneous or near simultaneous sequencing of thousands of nucleicacid molecules. High throughput sequencing is sometimes referred to as“next generation sequencing” or “massively parallel sequencing”.Platforms for high throughput sequencing include, without limitation,massively parallel signature sequencing (MPSS), Polony sequencing, 454pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, IonTorrent semiconductor sequencing, DNA nanoball sequencing, Heliscopesingle molecule sequencing, single molecule real time (SMRT) sequencing(PacBio), and nanopore DNA sequencing (e.g., Oxford Nanopore).

As used herein, the term “kit” includes a collection of items intendedfor use together. The items in the kit may or may not be in operativeconnection with each other. A kit can comprise, e.g., reagents, buffers,enzymes, antibodies and other compositions specific for the purpose. Akit can also include instructions for use and software for data analysisand interpretation. A kit can further comprise samples that serve asnormative standards. Typically, items in a kit are contained in primarycontainers, such as vials, tubes, bottles, boxes or bags. Separate itemscan be contained in their own, separate containers or in the samecontainer. Items in a kit, or primary containers of a kit, can beassembled into a secondary container, for example a box or a bag,optionally adapted for commercial sale, e.g., for shelving, or fortransport by a common carrier, such as mail or delivery service.

As used herein the term “substantially,” in phrases such as“substantially equal” or “substantially equimolar,” includesmeasurements or procedures in which, for example, two different samplesare found to have the same nucleic acid concentration, withinmeasurement error, or are produced by methods which should lead to thesame nucleic acid concentration (molar concentration) if reactionsproceed to completion. It will be appreciated that minor variations in,for example, concentration, are still within the meaning of“substantially equal,” for example, if such variations do not affect theuse of samples or interpretation of data from samples.

As used herein, the following meanings apply unless otherwise specified.The word “may” is used in a permissive sense (i.e., meaning having thepotential to), rather than the mandatory sense (i.e., meaning must). Thewords “include”, “including”, and “includes” and the like meanincluding, but not limited to. The singular forms “a,” “an,” and “the”include plural referents. Thus, for example, reference to “an element”includes a combination of two or more elements, notwithstanding use ofother terms and phrases for one or more elements, such as “one or more.”The term “or” is, unless indicated otherwise, non-exclusive, i.e.,encompassing both “and” and “or.”

II. Introduction

Provided herein are methods and compositions for manipulatingpolynucleotide samples during amplification, to provide equimolaramounts of polynucleotides in samples, or molar amounts that are relatedin a known ratio, or to normalize the polynucleotide concentrations invarious samples by using a detectable label when amplifying the samples.Each technique will be described in turn.

III. Preparation of Polynucleotides from Samples

For both methods, preparation of preparation of polynucleotides fromsamples are used. Methods of producing pooled, concentration adjusted(e.g., normalized) nucleic acid libraries involve providing librariesfrom a plurality of samples. Accordingly, while methods may describelibrary preparation with respect to a single sample, it is understoodthat the procedures can be used on a plurality of samples, for exampleserially or in parallel.

Referring to FIG. 1, library preparation begins with the provision ofpolynucleotides, i.e., DNA or RNA, from a sample. Polynucleotides can besourced from any biological sample. The biological sample can comprisepolynucleotides from the genome of a single individual or, in the caseof microbiome analysis, a plurality of organisms of different species.Samples used as source material include, without limitation, biologicalmaterials from an organism, cultured biological materials (e.g.,cultured cells), environmental samples (e.g., water soil or air) orforensic samples (e.g., blood, hair, semen). A biological sample from anorganism can comprise, for example, stool, blood, throat swab,nasopharyngeal swab, sputum, cerebral spinal fluid, serum, plasma, urineor a biopsy (e.g., tissue biopsy or liquid biopsy). In embodiments inwhich analysis of a subject individual's microbiome and/or microbialtranscriptome is desired, the sample can be one known to containmicroorganisms, e.g., stool.

Polynucleotides can be isolated from a sample by methods well known inthe art.

Polynucleotides can be extracted directly from the sample or cells inthe sample can first be lysed to release their polynucleotides. Nucleicacid in the sample can be treated with an RNA or DNA preservative, asappropriate, to inhibit degradation of nucleic acid. Polynucleotides canbe isolated from a sample by contacting the sample with a solid supportcomprising moieties that bind nucleic acids. For example, the solidsupport can be a column comprising silica or can comprise paramagneticsilica beads. After capturing nucleic acids in a sample the beads can beimmobilized with a magnet and impurities removed. DNA can be isolatedwith silica, cellulose, or other types of surfaces, e.g., Ampure SPRIbeads. Kits for such procedures are commercially available from, e.g.,Promega (Madison, Wis.) or Qiagen (Venlo, Netherlands). If the targetpolynucleotide is RNA the sample can be exposed to an agent thatdegrades DNA, for example, a DNase. For example, the Qiagen RNeasy kitcan be used to purify RNA.

Purified nucleic acids may be further fractionated to isolate targetsequences or to remove unwanted sequences. For example, in the case ofmicrobiome analysis involving analysis of microbiome RNA (e.g.,transcriptome), one may wish to eliminate noninformative RNA from thesample. Noninformative RNA can include, for example, host rRNA and aplurality of the most common host mRNA species in the sample. Forexample, in human blood, common RNA species include, for example,hemoglobin, myoglobin, 18S and 28S rRNA and 16S and 23S bacterial rRNA.To accomplish this, one can employ oligonucleotide probes that hybridizeto the species and that bear at least one extraction moiety (e.g., abiotin moiety).

Alternatively, one may wish to enrich for specific sequences by positiverather than negative selection. In certain embodiments preparation ofpolynucleotides can include sequence capture which can involvecontacting polynucleotides with oligonucleotide probes or baits attachedto solid supports. These probes capture polynucleotides having sequencesthat hybridize to the probe. Captured polynucleotides can be isolatedafter washing the solid support or supports to remove unboundpolynucleotides. This step may be performed after adapter ligation.

DNA can be chromosomal DNA or cell free DNA. Typically, chromosomal DNAis fragmented for processing into a library. For Illumina sequencing,nucleic acid fragments typically have a length between about 100 and1000 nucleotides.

Adapters can be attached to DNA molecules through ligation or throughprimer extension of primers comprising adapter sequences using DNAmolecules as a template.

Adapter ligation can involve blunt end ligation or overhang ligation. Inblunt end ligation in adapter with a blunt end is ligated to a DNAmolecule that also comprises a blunt end. In overhang ligation a DNAmolecule with an overhang, such as a “A” overhang or an overhangresulting from restriction endonuclease cleavage, is brought intocontact with an adapter molecule comprising a complementary overhang.

Polynucleotides subjected to fragmentation or cell free DNA typicallycomprise ends with single-stranded overhangs that require end repairbefore adapter ligation. End repair can be accomplished by, for example,an enzyme such as Klenow which cleaves back 5′ overhangs and fills in 3′overhangs. The result can be a blunt ended molecule or molecule with aspecific overhang.

Alternatively, target polynucleotides can be provided with adaptersthrough a primer extension reaction in which a primer molecule comprisesadapter sequences and a sequence that hybridizes to a location in atarget polynucleotide. For example, sequence-specific amplification cancomprise contacting a DNA sample with primers that hybridize tolocations flanking a target sequence. Primers can be extended such thatthe newly synthesized strand comprises both adapter sequences from theprimer and the target sequence upon second strand synthesis in theopposite direction the resulting polynucleotides will comprise a targetsequence flanked by adapter sequences. Accordingly, such amplificationcan comprise multiplex amplification in which a plurality of targetsequences is amplified simultaneously.

In the case of chromosomal DNA, the polynucleotides are typicallyfragmented. In the case of cfDNA, the molecules typically have a sizedistribution of about 140-180 nucleotides. In either case, molecules areend repaired, by Klenow, to produce either a blunt end or a singlenucleotide overhang, e.g., “A”.

A restriction site overhang DNA can be prepared for adapter ligation byend repair of polynucleotide molecules.

After at least one round of amplification, template molecules bearadapter-tags.

Purified nucleic acids may then be prepared for the methods disclosedherein.

IV. Methods and Compositions for Primer-Limited Amplification of NucleicAcids

A. Amplification

Provided herein are methods of providing equal or substantially equalmolar amounts of nucleic aids in a plurality of separate samples or in apooled sample from a plurality of separate samples, and/or of providingmolar amounts of nucleic acids in a plurality of separate sample, or ina pooled sample, that are related by known molar ratios. The methods ofthe first technique generally do not require labeling or reading oflabels and can be carried out in a few steps; typically, the methods donot require further procedures after nucleic acid amplification beyondcombining samples to pool them. Also provided herein are methods ofpreparing nucleic acid libraries comprising predetermined molar amountsof nucleic acid, which amounts have been determined, e.g., by methodsprovided herein. Also provided herein are pooled, nucleic acid librariescomprising normalized amounts of nucleic acid derived from each of aplurality of different samples, e.g., pooled libraries in which nucleicacids from different samples are present in equal molar amounts, orsubstantially equal molar amounts, and/or in amounts related to eachother in known molar ratios. Also provided herein are kits for use inproducing the nucleic acid libraries described herein.

Using the methods and compositions described herein, a pooled normalizednucleic acid sample may be produced from a plurality of individualsamples in procedures requiring nothing more than amplification ofnucleic acids in the individual samples and pooling of portions of theindividual samples. I.e., the methods and compositions described hereineliminate the necessity for labeling, reading labels, adjustingconcentrations, and the like, that are usually involved in producingnormalized libraries, thus eliminating time and costly reagents from theprocess.

The molar concentration or quantity of polynucleotides, e.g.,adapter-tagged polynucleotides, in the sample can be controlled bymethods described herein. Typically, the methods involve primer-inducedamplification of nucleic acids in a sample using a fixed molar amount ofprimer, and performing amplification through a sufficient number ofrounds that the primer is completely consumed; thus, the final molaramount of nucleic acid in the sample after the final round ofamplification will, in general, be related in a known manner to thefixed molar amount of primer, for example, equal or substantially equalto the molar amount of primer, though other possibilities are discussedherein. If a plurality of samples are amplified in this manner, and thesamples combined into a pooled sample, the molar amounts of nucleic acidfrom each sample in the pooled sample can be controlled by controllingthe relative amount (volume) of each individual sample used to producethe pooled sample. In certain embodiments, the amplification may occurafter individual samples are tagged with adapters and pooled; in theseembodiments, adapters used in different samples typically have differentprimer binding sites, and the molar amounts of amplified nucleic acidproduced from each sample is controlled by using fixed molar amount ofprimer for each of the different primer binding sites.

The methods and compositions provided herein are useful in both DNA andRNA amplification and quantification and/or normalization. Afteramplification and, e.g., pooling, nucleic acids in the sample may besequenced, using any suitable sequencing technology.

According to one method, nucleic acid in a sample is amplified in aplurality of primer extension reactions, using primers in a fixed molaramount. After sufficient rounds of amplification, the primer is used up,and the amount of amplified nucleic acid in the sample will be equal to,or substantially equal to, the fixed amount of primer used in theamplification, or will be related to the amount of primer used in aknown ratio. A plurality of separate samples may be amplified in thismanner, where the final amount of amplified nucleic acid in each sampleis known to be equal to, or substantially equal to, the fixed amount ofprimer used in the amplification in each sample, or related to theamount of primer used in a known ratio. The samples may then be pooled.The relative amount of nucleic acid from each sample that is included inthe pooled sample can be controlled by controlling the relative amount(volume) from each sample that is included the pooled sample.Alternatively, samples can be prepared separately with adaptersequences, where the adapter sequences for different samples beardifferent primer binding sites, then the samples can be pooled andprimer extension amplification performed on the pooled sample, where themolar amounts of nucleic acid for each sample are determined by themolar amount of primer used for that sample.

Any suitable method of nucleic acid amplification may be used,including, but not limited to, polymerase chain reaction (PCR)amplification, loop-mediated isothermal amplification (LAMP), reversetranscription loop-mediated isothermal amplification (RT-LAMP),strand-displacement amplification (SDA), helicase-dependentamplification (HDA), or transcription-mediated amplification (TMA). Forease of description, reactions will be discussed in terms of PCR;necessary adjustments for other methods of amplification will be readilyapparent to one of skill in the art.

In a nucleic acid amplification method, two primers may be used, e.g.,one for each strand of DNA, such as a forward primer and a reverseprimer. Nucleic acids in the sample may be prepared, for example, withadapter sequences that contain the primer binding sites for the firstprimer and the second primer (e.g., on different strands of DNA); incertain cases, the first and second primer binding sites are the same,and thus the first and second primers are the same; in other cases, thefirst and second primer binding sites are different, and thus the firstand second primers are different. In the former case, when a fixed molaramount of primer (which acts as both first and second primer) is addedand sufficient rounds of amplification performed, the molar amount ofamplified nucleic acid will be half that of the fixed molar amount ofprimer used, if the amplified nucleic acid is double stranded, or willbe equal to or substantially equal to the molar amount of primer used ifamplified nucleic acid is single-stranded. In the latter case, typicallyequal molar amounts of first and second primers are used and afteramplification the molar amount of amplified nucleic acid will be equalto or substantially equal to the molar amount of each primer used (asdouble stranded nucleic acid; if strands are melted or nucleic acid isonly single-stranded, e.g., RNA, then the molar amount will be equal todouble the molar amount of each primer). If one primer is used inexcess, amplification will continue after the limiting primer is used upfor double-stranded nucleic acid, and will stop when second primer isconsumed to produce single-stranded nucleic acid. For example, in a PCRamplification of double stranded DNA using a first primer for firststrand and a second primer for second strand, if first primer is presentin a molar amount of 0.1 mole, and second primer is present in a molaramount of 0.15 mole, exponential amplification will occur until the 0.1mole of first primer is consumed, and 0.1 mole of double-stranded DNAwill be produced. After that point, when strands are melted, only thesecond strand, with the second primer site, will continue to beamplified, with additional strands produced until the 0.15 mole ofsecond primer is consumed. Total molar amounts are 0.1 mole for firststrand and 0.15 mole for second strand. These amounts and amplificationtechnique (PCR) are merely exemplary, and amounts and ratios for otheramplification techniques will be apparent to one of skill in the art.For example, in certain amplification techniques an indefinite amount ofamplified nucleic acid may be produced at one or more steps, and molaramounts of final product may vary depending on conditions used; in suchcases, routine experimentation can show the proper conditions to controlthe reactions sufficiently that separate samples receive the sametreatment and produce amounts of nucleic acid in desired ratios.

Any suitable number of rounds of amplification (cycles) may be used,which may be dependent on the type of amplification procedure used. Incertain embodiments, e.g., in which PCR is the amplification method, thenumber of amplification rounds (cycles) is 2-50, or 2-40, or 2-30, or2-25, or 5-50, or 5-40, or 5-30, or 5-25, or 10-50, or 10-40, or 10-35,or 10-30, or 10-25, or 15-50, or 20-40, or 20-30, or 21-30, or 22-30, or23-30, or 24-30, or 25-30, or 26-30, or 27-30, or 28-30, or 21-29, or22-29, or 23-29, or 24-29, or 25-29, or 26-29, or 27-29, or 21-28, or22-28, or 23-28, or 24-28, or 25-28, or 26-28, or 21-27, or 22-27, or23-27, or 24-27, or 25-27; for example, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39 or 40 rounds of amplification (cycles).

In certain embodiments, a plurality of separate nucleic acid samples areamplified, using fixed molar amounts of primer in each sample. Nucleicacids in each sample may be prepared with adapter sequences withappropriate primer binding sites, as well as, e.g., barcodes (which maybe unique to each sample), sequencing platform-specific sequences, etc.The molar amount of primer in each sample may be the same or different.After amplification, the samples may be combined to produce a poolednucleic acid sample. The relative amounts of nucleic acid from eachseparate sample that are present in the pooled sample may be controlledby controlling the relative amount (volume) of each sample that isincorporated into the pooled sample. For example, separate samples canbe amplified using the same molar amount of primer in each sample, sothat after amplification, each sample contains the same or substantiallythe same molar amount of nucleic acid. If the entire volume of onesample is added to the pooled sample, and only one-tenth of the volumeof a second sample is added to the pooled sample, then the nucleic acidsfrom the first sample will be present in the pooled sample in 10:1 molarratio compared to the nucleic acids from the second sample. Any desiredratio of molar amounts of nucleic acids from separate samples may beproduced in the pooled sample by manipulating primer amounts in eachseparate sample used for amplification, or by manipulating relativeportions of each separate sample combined into the pooled sample, orboth.

The molar amount of primer added to each sample may be known, in whichcase the final molar amount of amplified nucleic acid in the sample canbe known, and the final molar amount of nucleic acid from that sample ina pooled sample can be known. Alternatively, for example, all or aportion of separate samples, in which primer sites are the same, may usethe same primer; a large central batch of primer solution may beprepared, and the relative molar amount of amplified nucleic acid inseparate samples amplified using the primer may be determined by therelative amount of the central primer solution added to the separatesamples. In this case, the actual molar amount of primer need not beknown; so long as each sample goes through sufficient rounds ofamplification to use all primer, the relative molar amounts of nucleicacid in each sample will be related according to the relative amount ofcentral primer solution used in each. For example, separate samplescontaining equal or substantially equal molar amounts of amplifiednucleic acids can be produced using the same volume of central primersolution in each. In certain embodiments, a known molar amount of primeris added to each sample, for example, with an exemplary volume forprimer addition of 50 μL, the concentration of the primer solution canbe 1-500 nanomolar (for a total amount of primer of 5×10⁻¹⁴ to 2.5×10⁻¹¹mole in a 50 μL portion), or 1-200 nanomolar, or 1-100 nanomolar, or2-80 nanomolar, or 5-70 nanomolar, or 8-60 nanomolar, or 10-50nanomolar, or 15-40 nanomolar, or 20-35 nanomolar, e.g., about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nanomolar (given an exemplaryvolume of 50 μL the molar amount may be calculated for each). The samemolar amount of primer may be added to each sample, or different, knownmolar amounts of primer, or amounts of primer related to each other inknown ratios, may be added to each sample. If a primer pair is used,molar amount can refer to the total of primer or to molar amount of eachmember of the pair.

In certain embodiments, it is desired to bring the amount of differentnucleic acids in a single sample, which may be present at very differentlevels, to the same or similar levels, or to levels where the differentnucleic acids may be detected. For example, a blood sample may containnucleic acid from two or more viruses, which are present in greatlydiffering amounts. If the sample is split into separate samples, each ofwhich is amplified with a fixed molar amount of primer (e.g., primerspecific for specific viral nucleic acid) through sufficient rounds touse all the primer, then, optionally, combining the amplified samples,viruses present at low levels relative to others can be readilydetected.

When separate samples are combined to form a pooled sample, any suitablenumber of separate samples may be uses, e.g., at least 2, 3, 4, 5, 10,20, 50, 100, 200, or 500 samples. In certain embodiments, a singlesample is split into 2 or more samples which, after amplification, canthen be combined into a pooled sample. When samples are pooled, incertain embodiments the pooled sample is treated (after pooling) toremove reaction components from amplification that are not necessary forfurther analysis and that may interfere with analysis, including but notlimited to unincorporated nucleotides, buffer, enzymes, and additives;cleanup protocols are well-known in the art.

In certain embodiments, genomic sequences are desired, and any suitablemethod for amplifying genomic nucleic acid may be used. In certainembodiments, it is desired to determine transcriptomes or other measuresof RNA, in which case any suitable method for amplifying RNA sequencesmay be used. If, e.g., PCR is to be used, RNA can be subject to firststrand synthesis (reverse transcription) to produce cDNA using firstprimers. In certain embodiments, the primers used during first strandsynthesis may comprise nucleotide sequences that are necessary and/orsufficient for sequencing on a specific sequencing platform. The cDNAmay include primer binding sites for one or more primers, which may beused in fixed molar amounts during amplification, as described herein.When performed on a plurality of different samples a pool of nucleicacid libraries can be produced in which the amounts of nucleic acidsfrom each library are normalized with respect to each other based atleast in part on the amounts primer used for amplification in eachsample. The pooled sample can be used to populate a sequencing flowcell. Upon sequencing, quantities of nucleic acid sequences betweensamples can be compared directly to one another.

Also provided here are normalized nucleic acid libraries, e.g., DNAlibraries and pooled, normalized nucleic acid libraries, e.g., DNAlibraries in which nucleic acids from different samples are present inknown molar ratios to each other.

Using the methods and compositions described herein, any suitable numberof different samples may be amplified to provide final concentrations ofnucleic acid in the samples that are intended to be equal orsubstantially equal. It will be appreciated that real-world conditionscan cause amplified samples to be at nucleic acid concentrations thatare different from the intended concentration; however, using thedescribed methods and compositions it is possible to obtain a relativelynarrow range of concentrations. This allows a greater number of samplesto be analyzed on an analyzer that takes in a mixture of samples, e.g.,a sequencer such as an Illumina sequencer, and remain within qualitycontrol (QC) limits. Any suitable range may be set as the QC limit, suchas within 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10% of the mean; and aplurality of samples amplified by the methods and compositions of theinvention can be within the desired QC range, such as at least 20, 30,40, 50, 55, 60, 65, 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99% of the samples, for example, at least 70%, such as at least80% or at least 90%. See Example 3, in which the QC limit for asequencer was set at within 50% of the mean number of reads per sample,and 93.4% of the samples fell within the QC limit. It will beappreciated that this is merely exemplary, and that any suitable QClimit may be set, with corresponding percentage of samples fallingwithin the limit.

B. Preparation of Nucleic Acid Libraries

Preparation of nucleic acid libraries comprising polynucleotides inknown molar ratios can involve creation of adapter taggedpolynucleotides. Adapters can be provided by hybridization of primerscomprising adapter sequences to target molecules, followed by primerextension, or by ligation of the adapters to nucleic acids in a sample.Primer extension is typically used with single-stranded target nucleicacids. Double stranded nucleic acids can be tagged through primerextension or ligation. Primer extension can be useful to amplify targetsequences in long nucleic acid molecules. Ligation of adapter isparticularly useful for tagging short, double stranded nucleic acidmolecules, such as cell-free DNA.

Methods disclosed herein comprise performing a plurality of primerextension reactions on nucleic acid molecules in a sample, eitheroriginal, untagged molecules, or adapter-tagged molecules. Primerextension is performed using polynucleotide primers. Primer extensioncan use a single set of primers or a plurality (e.g., two) primer sets.In the case of a single primer set, all or substantially all of theprimer can be used in the primer extension reactions. In the case ofmultiple primer sets, some or all of the different primers can be used.

The product of primer extension reactions is a nucleic acid library, inwhich the molar amount of nucleic acids in the library is determined bythe molar amount of primer used. This can provide an indication of theamount of nucleic acid in a single library, or the relative amounts ofnucleic acids in a plurality of libraries.

In order to quantify nucleic acids in a sample or a library, a knownmolar amount of primer can be used during amplification. The molaramount of amplified nucleic acid will be related in a known manner tothe known molar amount of primer used.

In certain embodiments of the disclosed methods, adapter-taggedmolecules comprise sequencing platform-specific sequences compatiblewith a particular sequencing platform. For example, in the case ofIllumina MiSeq devices, platform-specific sequences can include asequencing primer binding site and a flow cell cluster primer bindingsite. Preparation of tagged libraries whose molar concentration can beknown is not limited to any one technology. For example, this method canbe used to prepare PacBio, Ion Torrent, and Oxford Nanopore sequencinglibraries. In case of PacBio, the SMRT bells can be tagged on one orboth side of the library fragments. In case consensus sequencing isdesired, where the sequencing proceeds through the SMRT bells, the tagcan be connected to the library fragment via a cleavable linker, andwould be released prior to sequencing. Ion Torrent sequencing technologyutilizes adapters very similar to Illumina, enabling on-bead clonalamplification of library fragments. These adapters can also be taggedwith a quantifiable tag that can be used to easily quantify the molarconcentration of the library fragments prior to clonal amplification, orprior to sequencing.

1. Preparing RNA for Amplification

The methods and compositions can be used for samples comprisingsingle-stranded nucleic acids, in particular, RNA. In one embodiment, acDNA library is produced from RNA in which molecules of first strandsynthesis bear sequencing-platform specific sequences necessary and/orsufficient for sequencing on the sequencing platform. It will beunderstood that the original template can be DNA taken from any source,rather than RNA.

Referring to FIG. 2, a sample comprising RNA molecules is provided. Afirst or forward primer/adapter is provided comprising a sequence forbinding to an RNA molecule, for example, a degenerate hexamer sequence(hex). In certain embodiments, the primer further includes sequencingplatform-specific sequences. In this example, platform-specificsequences are designated PSA1 and PSA2. For example, in the case ofIllumina sequencers, these sequences may have the P5 and P7 sequences.Typically, adapters will also comprise a sample barcode (designated inFIG. 2 as (“SB1” and “SB2”)). In the example of FIG. 2, theadapter/primers also bear primer binding sites 1 and 2. In Illuminasequencers, these sites may be used as sequencing primer binding sites.

The primer/adapter is hybridized to the RNA. Using a reversetranscriptase, such as Murine Maloney leukemia virus reversetranscriptase, the primer/adapter is extended, producing a cDNA strandhaving, at the 3′ end, a poly C tail. A second adapter/primer is addedto function as a further template. In this example, the secondadapter/primer comprises, at the 3′ end, a poly G tail adapted forhybridizing with the poly C tail. In certain embodiments the secondadapter/primer also comprises sequencing platform-specific sequencesand/or sample barcode sequences. The polymerase further extends thecDNA, producing a first strand adapter-tagged cDNA molecule comprising acDNA insert flanked by adapter sequences. In the example of FIG. 2, theadapter sequences include platform-specific adapter sequences PSA1 andPSA2′ (a template for PSA2 on complementary strand), sample barcodes SB1and SB2′ (a template for sample barcode SB2 on complementary strand),and secondary primer binding sites PBS1 and PBS2′ (a template for PBS2primer binding site on complementary strand).

The product of this reaction is adapter-tagged cDNA molecules.

2. Amplification of Adapter-Tagged Nucleic Acids

Adapter-tagged polynucleotides can be amplified in a plurality or roundsof primer extension, for example, PCR. At least one adapter-tag cancomprise a sample nucleotide barcode, one or more primer binding sitesand, optionally, sequencing platform-specific sequences for engagingamplified polynucleotides to a sequencing platform.

Amplification of adapter-tagged polynucleotides in each sample proceedsby contacting the adapter-tagged polynucleotides with primers. Theprimers are present in fixed molar amount, so that the final molaramount of amplified polynucleotide is determined by the molar amount ofthe primers.

In certain embodiments, forward and reverse primer binding sites are thesame, and a single primer set comprising primers is used foramplification. In certain embodiments, amplification will proceed fromforward and reverse primer binding sites having different sequences andflanking a region to be amplified. In this case, primers may be providedas primer pairs, a forward primer and a reverse primer, each member ofthe pair binding to one of the different binding sites. In such asituation the primers may be present in equal molar amount, or oneprimer may be present in known molar excess to the other.

Referring to FIG. 3, according to one amplification strategy,adapter-tagged cDNA molecules are prepared from RNA by the methoddescribed above, except that adapter/primer molecules may bear only aset of forward and reverse primer binding sites (designated here as PBS1and PBS2′). The result may be an adapter-tagged nucleic acid insertflanked by primer binding sequences.

Referring to FIG. 4, in this example, the adapter-tagged polynucleotideis a single stranded DNA molecule. (For purposes of clarity, thecomplement of this strand is also shown, however, a double strandedadaptor-tagged polynucleotide could be used in the same manner.) Theadapter-tagged polynucleotide comprises a first primer binding site(PBS1), a target nucleic acid insert (in this case cDNA) and thecomplement of a second primer binding site (PBS2′). These molecules arecontacted with two sets of primers.

Primers in a first set can comprise a forward primer comprising bindingsequence (PBS1) that binds to first primer binding sites (PBS1′) on thecomplement of the adapter-tagged polynucleotide. They can furthercomprise second primer binding sites which may function asplatform-specific adapter sequences (PSA1). Primers in the first set mayalso comprise a sample barcode (SB1). Primers in the first set alsoinclude a reverse primer comprising a primer binding sequence (PBS2)that binds to second primer binding sites (PBS2′) on the adapter-taggedpolynucleotide. They can further comprise second primer binding siteswhich may function as platform-specific adapter sequences (PSA2).Primers in the first set may also comprise a second sample barcode(SB2).

Primers in a second set can comprise a sequence that binds to a primerbinding site on the first set of primers. In the example depicted inFIG. 4, these primers bear primer binding sequences designated PSA1 andPSA2. These primers may not bear sequences contained in the first set ofprimers. For example, they may be shorter than the first set of primers.They may not bear sample barcodes. They may not bear sequenceshybridizing to the primer binding sites on the initial adapter-taggedpolynucleotide. The members of the second primer pair are added in afixed molar amount, e.g., in equal molar amounts, or in a known ratio ofmolar amounts (one primer in excess).

In certain embodiments of the strategy, primer binding site 1 may alsoserve as a sequencing primer in a high throughput sequencing system,such as MiSeq, and/or the platform-specific adapter sequence may serveas a flow cell cluster site sequence, e.g., P5 and P7.

Upon amplification, the first set of primers convert adapter-taggednucleic acid molecules into longer adapter-tagged nucleic acid moleculesthat comprise a sample barcode and the second primer binding site. Infurther rounds of amplification, primers of the second set will be usedfor strand extension for the longer adapter-tagged nucleic acidmolecules. As a result, after sufficient rounds of amplification thepopulation of amplified polynucleotides will reach a limit based on themolar amounts of the primers in the second pair of primers. If theprimers are present in equal molar amounts, the final amplified molaramount of double-stranded nucleic acid will be equal to or substantiallyequal to the molar amount of each of the primers.

According to another amplification strategy, the original molecules aresingle- or double-stranded polynucleotides (e.g., end-repairedmolecules). To these molecules are ligated adapters having, at least,primer binding sites. In the case of so-called “Y”-shaped adapters, theprimer binding site on the free 5′ end of the adapter is different thanthe primer binding site on the free 3′ end of the adapter. Suchmolecules can be amplified with primer sets that hybridize to the primerbinding sites and that may comprise platform-specific adapter sequencesand, optionally, sample barcode sequences. The members of the primer setare added in fixed molar amounts, for example, in equal molar amounts,or in molar amounts related to each other in a known ratio. Aftersufficient rounds of amplification, amplified molecules are producedwhose molar amount is determined by the molar amounts of primers added.

According to another amplification strategy, adapter-tagged nucleic acidmolecules are amplified with a single primer set added in fixed molaramounts. After enough rounds of amplification to consume the primers,amplified nucleic acids comprising nucleic acid insert flanked byadapter sequences comprising primer binding sites and sample barcodeswill be present in molar amounts determined by the molar amounts ofprimer added.

After primer extension and/or amplification, unincorporated primers,that is, primers that have not been used to support primer extension aspart of strand synthesis, are typically removed from the sample; in thepresent methods, such a step may be unnecessary since all primer shouldbe incorporated. However, if desired, potential unincorporated primerscan be separated from the nucleic acid library by, for example, sizeselection or by hybridization to some complementary probes attached to asolid support.

C. Nucleic Acid Libraries

Using the methods and compositions described herein, it is possible toproduce nucleic acid libraries.

In certain embodiments, the invention provides a set of a plurality ofseparate nucleic acid libraries, where each library contains amplifiedpolynucleotides and where the molar amount of polynucleotides in eachlibrary are the same or substantially the same, or are related as aknown ratio to each other. Each library may be in a separate container.The amplified polynucleotides may contain non-natural sequences, e.g.,barcodes, primer sites, sequencing platform-specific sequences, and thelike.

In certain embodiments the invention provides a pooled nucleic acidlibrary including amplified nucleic acids from a plurality of differentsamples, where the nucleic acids from each sample are present in equalor substantially equal molar amounts, or in molar amounts that are knownratios of each other, and where the nucleic acids do not include alabel. Nucleic acids from each sample may also incorporate a barcodethat is different for each different sample.

D. Quantifying Amounts of Nucleic Acids in Each of a Plurality ofSamples and Preparing Normalized and Normalized, Pooled Libraries

Nucleic acid molecules in nucleic acid libraries so produced can bequantified based on the molar amounts of primers used in amplification.The molar amount of amplified nucleic acids in one library can berelated to the molar amount of amplified nucleic acid in another librarybased on the molar amounts of primer used to produce each library. Forexample, if equal molar amounts of primer are used to produce amplifiednucleic acids in each library, then the final molar amount of amplifiednucleic acid in each library will be equal or substantially equal.

It will be appreciated that the precise molar amount of primer used toproduce each library need not be known in order to produce libraries ofamplified nucleic acids that are present in the same molar amount ineach library, or in molar amounts related to each other in a knownratio. For example, if all samples used to produce nucleic acidlibraries are tagged with adapters with the same primer bindingsequences, a master pool of primers specific for the primer bindingsequences may be prepared, and known volumes of the pool added to eachsample. If equal volumes are added, equal molar amounts of primer areused and equal, or substantial equal, final molar amounts of amplifiednucleic acid will be produced in each sample. If different volumes areused, the final molar amounts of amplified nucleic in the samples willbe related according to the volumes of primers used in the samples.

Generally, no labeling or measurements of label or other properties ofthe nucleic acids is needed to quantify the final molar amount ofnucleic acid in separate amplified libraries or in pooled libraries,since the final molar amount is directly related to the molar amount ofprimer used to produce the amplified nucleic acids. In certainembodiments, quantitation does not comprise providing an intercalatingdye to nucleic acids and measuring an amount of dye intercalated. Incertain embodiments, quantitation does not comprise providing labeledprobes that hybridize to nucleic acid molecules and measuring an amountof probe hybridized to the nucleic acids.

E. Production of Libraries with Adjusted/Normalized Amounts ofPolynucleotides

Using these methods, the relative molar amount of amplified nucleicacids in a sample can be controlled; in certain embodiments, theabsolute molar amount of amplified nucleic acids in a sample is known. Aplurality of libraries can have known amounts or concentrations ofnucleic acid, and/or have amounts of nucleic acid in known molar ratios.These libraries can be used to produce pooled libraries.

In one embodiment, individual amplified nucleic acid libraries areproduced by, for example, amplifying nucleic acids with fixed molaramounts of primers. In some cases, the molar amount of primer will bethe same for all individual libraries. Then, portions of each amplifiedlibrary can be combined into a pool. The molar amounts of primers usedto produce each library, together with the portion of each library addedto the pool, determines the relative molar amount of each library'snucleic acids in the final pool. For example, if equal molar amounts ofprimer are used in amplification to produce each library, and if 50% ofeach library is pooled with others to produce a pooled library, thennucleic acids from each individual library will be present in the pooledlibrary in equal or substantially equal molar amounts (and, since thereis only one volume for the pooled library, as equimolar or substantiallyequimolar concentrations). It will be appreciated that by manipulatingone or both of molar amount of primer and portion of sample added tofinal pool, virtually any desired ratio of molar amounts of nucleicacids from the various libraries may be produced in the final pooledlibrary. If the absolute molar amount of primers used to produce one ormore individual amplified libraries is known, then the absolute molaramount of nucleic acid from those libraries in the pooled sample can becalculated.

In subsequent steps as described herein, nucleic acid libraries in whichamounts of nucleic acid have been determined, or pooled libraries inwhich molar amounts of nucleic acids from individual samples are known,or are related in known molar ratios, can now be sequenced. Sequencingcan proceed, for example, by high throughput sequencing.

F. Sequencing Libraries

Sequencing nucleic acid libraries produces sequence reads of thepolynucleotides sequenced. Because nucleic acids in each nucleic acidlibrary can bear a sample barcode sequence reads can be sorted into binsbased on the original library from which they are sourced. Sequencereads from individual libraries can be subject to further analysis. Inone embodiment, redundant sequences can be collapsed into an originalsequence, e.g., a nucleotide by nucleotide. Raw sequence reads orcollapsed reads may be referred to herein as “sequenced nucleic acids”.Sequenced nucleic acids in any library can be analyzed to determinequantities of target sequences in the sample. For example, if thelibrary comprises sequences of a microbiome, sequenced nucleic acids canbe analyzed to determine species present in the sample and amount ofeach species. As another example, if the library comprises sequences ofa transcriptome, sequenced nucleic acids can be analyzed to determinemRNAs present in the sample and amount of each mRNA.

There are many bioinformatics methods that convert raw sequences intosecondary data. For example: Taxonomy classification uses databases withunique sequences belonging to different organisms. Once a sequence ismatched to the database, the presence of a specific organism can bedetected. By counting the sequences used to identify each organism,their relative abundances can also be measured. Functional assignmentscan also be made from the sequence reads. A database that correlatessequences to functions is used to convert sequencing reads intobiochemical functions.

FIG. 5 shows one exemplary protocol for amplification followed bysequencing.

G. Kits

Also provided herein are kits. Kits can comprise adapters and/or primersas disclosed herein. Kits can comprise reagents for performingbiochemical reactions, such as reagents for primer extension and/or foramplification, such as PCR. Such reagents can include any ofpolymerases, reverse transcriptases, nucleotides and buffers, and otherreagents and substances necessary or useful in a particularamplification technique. Kits can comprise containers for containingcompositions of matter. Kits can comprise containers comprising theaforementioned containers. Kits also can comprise instructions for use,e.g., printed instructions.

In certain embodiments, kits comprise sets of primers as describedherein, where each set of primers is contained in a separate containeror containers, for example, a set of first and second primers, such asforward and reverse primers. Each container has a fixed molar amount ofprimer which is in a known ratio to the molar amounts of primer in othercontainers, e.g., an equal molar amount or substantially equal molaramount. The absolute molar amount of primer in one or more containersmay be known. Primers from the separate containers may be used in primerextension amplification in separate samples, to produce amplifiednucleic acids in the samples. The first and second primers may have thesame or different polynucleotide sequences. The kit can further includeadapter sequences, which may include primer binding sites, barcodes,sequencing platform-specific sequences, or a combination thereof. Thekit may further include reagents for performing nucleic acidamplification, for example, reagents for performing PCR, LAMP, RT-LAMP,SDA, HDA, or TMA.

A kit may also contain a single container with pooled primers, which areadapted for binding to universal primer binding sites provided byadapters, where a volume of the pooled primers added to each of separatenucleic acid samples determines the final molar ratio of amplifiednucleic acids in the samples after amplification. The absolute molaramount of primer in a volume of the pooled primer may be known. Adaptersmay also be packaged in the, or in a separate kit. A single containermay also contain an adapter, aliquots of which may be used in individualsamples to provide adapter-modified nucleic acids ready foramplification; however, in general, adapter may have barcode sequencesto distinguish separate nucleic acid samples, and adapters withdifferent barcodes are packaged in different containers. Adapters mayinclude primer binding sites (e.g., the same primer binding site will bepresent in each separate sample after incorporation of adapters intonucleic acids in the sample), barcodes, sequencing platform-specificsequences, or a combination thereof. The kit may further includereagents for performing nucleic acid amplification, for example,reagents for performing PCR, LAMP, RT-LAMP, SDA, HDA, or TMA.

V. Methods and Compositions for Amplification of Nucleic Acids withDetectable Labels

Also provided herein are methods of determining molar concentration oramounts of nucleic acid molecules in a sample. Further provided hereinare methods of preparing normalized nucleic acid libraries comprisingpredetermined molar amounts of nucleic acid, which amounts have beendetermined, e.g., by methods provided herein. Also provided herein arepooled, nucleic acid libraries comprising normalized amounts of nucleicacid derived from each of a plurality of different samples. That is,each library contributes a normalized amount of nucleic acid moleculesto the pooled library. In certain embodiments, the normalized librarieshave equal molar amounts of nucleic acid (that is, number of moleculesas opposed to mass of nucleic acid).

The molar concentration or quantity of adapter-tagged polynucleotides inthe sample can be determined. Normalized, pooled nucleic acid librariescan be prepared and sequenced directly, e.g., without furtheramplification or with limited amplification, e.g., fewer than any of aone, 2, 5, or 10, rounds of amplification.

Methods of determining amounts of nucleic acid in a sample compriseperforming one or a plurality of primer extension reactions usingprimers, at least a portion of which comprise a detectable label, anddetecting label incorporated into synthesized molecules. In certainembodiments, nucleic acids are quantified after one or two rounds ofprimer extension using labeled primers, e.g., after first and/or secondstrand synthesis from a template molecule, such as RNA. In otherembodiments, nucleic acids are quantified after a plurality of rounds ofamplification, e.g., after a plurality of rounds of PCR, in which atleast a portion of the primers used for amplification comprise adetectable label.

According to one method an amount of RNA in a sample is determined. TheRNA can be subject to first strand synthesis (reverse transcription) toproduce cDNA using first primers, at least a portion of which comprise adetectable label, such as a fluorescent tag. The amount of detectablelabel incorporated into the first strand synthesis product can bemeasured. The amount of nucleic acid in the sample is a function ofsignal produced by the detectable label. In certain embodiments, theprimers used during first strand synthesis may comprise nucleotidesequences that are necessary and/or sufficient for sequencing on aspecific sequencing platform. When performed on a plurality of differentsamples a pool of nucleic acid libraries can be produced in which theamounts of nucleic acids from each library are normalized with respectto each other based at least in part on the amounts of nucleic aciddetected in each sample. The pooled sample can be used to populate asequencing flow cell. Upon sequencing, quantities of nucleic acidsequences between samples can be compared directly to one another.

According to one method, nucleic acid in a sample is amplified, forexample by a plurality of rounds of PCR, using primers bearing adetectable label. After amplification, the amount of amplified nucleicacid in the sample is measured as a function of incorporated detectablelabel. Pooled, normalized nucleic acid libraries can be prepared from aplurality of samples based on the amounts of nucleic acid in each sampleas determined by the method provided herein.

Also provided here are normalized DNA libraries and pooled, normalizedDNA libraries in which at least some of the amplified molecules bear thedetectable label.

A. Preparation of Nucleic Acid Libraries Comprising PolynucleotidesComprising a Label.

Preparation of nucleic acid libraries comprising polynucleotidescomprising a detectable label typically involves creation of adaptertagged polynucleotides. Adapters can be provided by hybridization ofprimers comprising adapter sequences to target molecules, followed byprimer extension, or by ligation of the adapters to nucleic acids in asample. Primer extension is typically used with single-stranded targetnucleic acids. Double stranded nucleic acids can be tagged throughprimer extension or ligation. Primer extension can be useful to amplifytarget sequences in long nucleic acid molecules. Ligation of adapter isparticularly useful for tagging short, double stranded nucleic acidmolecules, such as cell-free DNA. Detectable label is typicallyintroduced during a primer extension step, which can be performed on atarget molecule or during amplification of double stranded molecules.

Methods disclosed herein comprise performing one or a plurality ofprimer extension reactions on nucleic acid molecules in a sample, eitheroriginal, untagged molecules, or adapter-tagged molecules. Primerextension is performed using polynucleotide primers, at least a portionof which bear a detectable label. Primer extension can use a single setof primers or a plurality (e.g., two) primer sets. In the case of asingle primer set, all or substantially all of the primers can bear alabel or a portion or a fraction (fewer than 100%) can bear a label. Inthe case of multiple primer sets, all of the primers can bear a label.More typically, a portion (fewer than all) primers can bear a detectablelabel. In such embodiments, a first set of primers can comprise primersbearing a label while primers in a second set of primers do not bear alabel. For example, all or substantially all primers in a first set canbear a label while primers in a second set bear no label. In anotherembodiment, a portion of the primers in each a plurality of primer setsbear a label.

The ratio of primer or adapter bearing a detectable label to those notbearing a detectable label can range from 1:100 to 100:1. Moretypically, the ratio will be between about 10:1 to 1:10, about 1:5 to5:1, about 1:3 to 3:1, about 1:2 to 2:1 or about 1:1. In certainembodiments all of the primers (100%) bear a detectable label. Theseratios apply where two sets of primers are used, each binding to adifferent primer binding site, and primers in one set all bear a label,while no primers in the other set bear a label.

Depending on the primer strategy used and percent of primers bearing adetectable label, the collection of amplified molecules can includethose bearing no label, those bearing a single label and those bearingtwo labels. The percentage of amplified molecules falling in each groupdepends on the ratio of labeled two unlabeled primers used. Where thepercentage of labeled primers is “p”, the percentage of amplifiedmolecules bearing to labels will be p² P squared. The percentage ofamplified molecules bearing one label will be 2*p*(1−p). The percentageof amplified molecules bearing no label will be (1−p)².

The product of primer extension reactions is a nucleic acid library, inwhich at least a portion of the polynucleotides in the libraryincorporate the detectable label. The amount of detectable label in thelibrary can be measured. This provides an indication of the amount ofnucleic acid in a single library, or the relative amounts of nucleicacids in a plurality of libraries.

In order to quantify nucleic acids in a sample or a library, detectablelabels are incorporated into nucleic acids in a stoichiometric mannerduring primer extension or amplification. The amount of detectable labelincorporated provides a measure of the molar concentration of nucleicacids in the sample.

In certain embodiments incorporated detectable label can be measuredafter a single primer extension reaction using extension primers bearinga detectable label. In other embodiments detectable label can bemeasured after a plurality of rounds of primer extension, e.g., after aplurality of rounds of amplification for example by PCR.

In certain embodiments of the disclosed methods, adapter-taggedmolecules comprise sequencing platform-specific sequences compatiblewith a particular sequencing platform. For example, in the case ofIllumina MiSeq devices, platform-specific sequences can include asequencing primer binding site and a flow cell cluster primer bindingsite. Preparation of tagged libraries whose molar concentration can beeasily determined is not limited to any one technology. For example,this method can be used to prepare PacBio, Ion Torrent, and OxfordNanopore sequencing libraries. In case of PacBio, the SMRT bells can betagged on one or both side of the library fragments. In case consensussequencing is desired, where the sequencing proceeds through the SMRTbells, the tag can be connected to the library fragment via a cleavablelinker, and would be released prior to sequencing. Ion Torrentsequencing technology utilizes adapters very similar to Illumina,enabling on-bead clonal amplification of library fragments. Theseadapters can also be tagged with a quantifiable tag that can be used toeasily quantify the molar concentration of the library fragments priorto clonal amplification, or prior to sequencing.

B. Libraries for Quantification and Sequencing After One PrimerExtension Reaction

In one embodiment, incorporated detectable label is measured after one,or a few primer extension reactions. The method can be used for samplescomprising single-stranded nucleic acids, in particular, RNA. In oneembodiment of the strategy, a cDNA library is produced from RNA in whichmolecules of first strand synthesis bear sequencing-platform specificsequences necessary and/or sufficient for sequencing on the sequencingplatform. At least a portion of these molecules incorporated detectablelabel. It will be understood that the original template can be DNA takenfrom any source, rather than RNA.

Referring to FIG. 6, a sample comprising RNA molecules is provided. Afirst or forward primer/adapter is provided comprising a sequence forbinding to an RNA molecule, for example, a degenerate hexamer sequence(hex). In certain embodiments, the primer further includes sequencingplatform-specific sequences. In this example, platform-specificsequences are designated PBS1 and PBS2. For example, in the case ofIllumina sequencers, these sequences may have the P5 and P7 sequences.Typically, adapters will also comprise a sample barcode (designated inFIG. 2 as (“SB1” and “SB2”)). In the example of FIG. 6, theadapter/primers also bear primer binding sites 1 and 2. In Illuminasequencers, these sites may be used as sequencing primer binding sites.Some or all of the first primer/adapter molecules comprise a detectablelabel (*).

The primer/adapter is hybridized to the RNA. Using a reversetranscriptase, such as Murine Maloney leukemia virus reversetranscriptase, the primer/adapter is extended, producing a cDNA strandhaving, at the 3′ end, a poly C tail. A second adapter/primer is addedto function as a further template. In this example, the secondadapter/primer comprises, at the 3′ end, a poly G tail adapted forhybridizing with the poly C tail. In certain embodiments the secondadapter/primer also comprises sequencing platform-specific sequencesand/or sample barcode sequences. The polymerase further extends thecDNA, producing a first strand adapter-tagged cDNA molecule comprising acDNA insert flanked by adapter sequences. In the example of FIG. 6, theadapter sequences include platform-specific adapter sequences PSA1 andPSA2′, sample barcodes SB1 and SB2′, and secondary primer binding sitesPBS1 and PBS2′, at least one primer binding site. Typically, librarymolecules are separated from unincorporated primers to remove excesslabel.

The product of this reaction is adapter-tagged cDNA molecules, at leastsome of which bear a detectable label.

In certain embodiments the nucleic acid libraries can be sequencedwithout performing subsequent amplification reactions, with no more than2, 3 or 4 rounds of amplification or with more than 2, 4, 8 or 16 roundsof amplification.

C. Libraries for Quantification and Sequencing After Amplification ofAdapter-Tagged Nucleic Acids

Detectable label can be incorporated into adapter-tagged polynucleotidesby one or a plurality or rounds of primer extension, for example,amplification, for example, PCR. At least one adapter-tag can comprise asample nucleotide barcode, one or more primer binding sites and,optionally, sequencing platform-specific sequences for engagingamplified polynucleotides to the sequencing platform.

Amplification of adapter-tagged polynucleotides in each sample proceedsby contacting the adapter-tagged polynucleotides with primers. At leasta portion of the primers bear a detectable label.

In certain embodiments, forward and reverse primer binding sites are thesame, and a single primer set comprising labeled primers is used foramplification and incorporation of detectable label. In certainembodiments, amplification will proceed from forward and reverse primerbinding sites having different sequences and flanking a region to beamplified. In this case, primers may be provided as primer pairs, aforward primer and a reverse primer, each member of the pair binding toone of the different binding sites. In such a situation one or both ornone of the members of the pair may bear a detectable label.

Referring to FIG. 3, according to one amplification strategy,adapter-tagged cDNA molecules are prepared from RNA by the methoddescribed above, with the following differences. First, theprimer/adapters used to first incorporate adapter sequences intomolecules may not bear a detectable label. Second, the adapter/primermolecules may bear only a set of forward and reverse primer bindingsites (designated here as PBS1 and PBS2′). The result may be anadapter-tagged nucleic acid insert flanked by primer binding sequences.

Referring to FIG. 7, in this example, the adapter-tagged polynucleotideis a single stranded DNA molecule. (For purposes of clarity, thecomplement of this strand is also shown, however, a double strandedadaptor-tagged polynucleotide could be used in the same manner.) Theadapter-tagged polynucleotide comprises a first primer binding site(PBS1), a target nucleic acid insert (in this case cDNA) and a secondprimer binding site (PBS2′). These molecules are contacted with two setsof primers.

Primers in a first set can comprise a forward primer comprising bindingsequence (PBS1) that binds to first primer binding sites (PBS1′) on thecomplement of the adapter-tagged polynucleotide. They can furthercomprise second primer binding sites which may function asplatform-specific adapter sequences (PSA1). Primers in the first set mayalso comprise a sample barcode (SB1). Primers in the first set alsoinclude a reverse primer comprising a primer binding sequence (PBS2)that binds to second primer binding sites (PBS2′) on the adapter-taggedpolynucleotide. They can further comprise second primer binding siteswhich may function as platform-specific adapter sequences (PSA2).Primers in the first set may also comprise a second sample barcode(SB2). In this example, primers in the first set are free of adetectable label. Alternatively, a portion of them may comprise adetectable label.

Primers in a second set can comprise a sequence that binds to a primerbinding site on the first set of primers. In the example depicted inFIG. 7, these primers bear primer binding sequences designated PSA1 andPSA2. These primers may not bear sequences contained in the first set ofprimers. For example, they may be shorter than the first set of primers.They may not bear sample barcodes. They may not bear sequenceshybridizing to the primer binding sites on the initial adapter-taggedpolynucleotide. Typically, a portion, or all, of the primers in thesecond set comprise a detectable label (*). In certain embodiments, onlyone of the members of the pair of second primers bears the detectablelabel. In the embodiment shown here, both members of the pair of secondprimers bears the detectable label.

In certain embodiments of the strategy, primer binding site 1 may alsoserve as a sequencing primer in a high throughput sequencing system,such as MiSeq, and/or the platform-specific adapter sequence may serveas a flow cell cluster site sequence, e.g., P5 and P7.

Upon amplification, the first set of primers convert adapter-taggednucleic acid molecules into longer adapter-tagged nucleic acid moleculesthat comprise a sample barcode and the second primer binding site. Infurther rounds of amplification, primers of the second set comprisingthe detectable label will be used for strand extension for at least someof the longer adapter-tagged nucleic acid molecules. As a result, afterseveral rounds of amplification the population of amplifiedpolynucleotides will include a first subpopulation bearing thedetectable label and a second subpopulation free of the detectablelabel. If the ratio of second primers to first primers is high, and thesecond primers, but not the first bear a detectable label, a majority orsubstantially all of the amplified molecules will bear a detectablelabel.

According to another amplification strategy, the original molecules aresingle- or double-stranded polynucleotides (e.g., end-repairedmolecules). To these molecules are ligated to adapters having, at least,primer binding sites. In the case of so-called “Y”-shaped adapters, theprimer binding site on the free 5′ end of the adapter is different thanthe primer binding site on the free 3′ end of the adapter. Suchmolecules can be amplified with primer sets that hybridize to the primerbinding sites and that comprise platform-specific adapter sequences and,optionally, sample barcode sequences. At least a portion of the primersin this amplification set are detectably labeled. After several roundsof amplification, amplified molecules, at least some of which bear adetectable label, are produced.

According to another amplification strategy, adapter-tagged nucleic acidmolecules are amplified with a single primer set in which a fraction,but not all, of the primers in the set bear a detectable label. Afterseveral rounds of amplification amplified nucleic acids comprise nucleicacid insert flanked by adapter sequences comprising primer binding sitesand sample barcodes. In this population a percentage of molecules willbear no detectable labels a percentage of molecules will bear onedetectable label (on either side of the molecule) and a certainpercentage of molecules will bear to detectable labels, one on eachstrand.

After primer extension and/or amplification, unincorporated primers,that is, primers that have not been used to support primer extension aspart of strand synthesis, are typically removed from the sample.Unincorporated primers can be separated from the nucleic acid libraryby, for example, size selection or by hybridization to somecomplementary probes attached to a solid support.

D. Quantifying Amounts of Nucleic Acids in Each of a Plurality ofSamples and Preparing Normalized and Normalized, Pooled Libraries

Nucleic acid molecules in nucleic acid libraries so produced can bequantified by measuring detectable label incorporated into librarymolecules. Determining amounts can be relative or absolute. Quantifyingrelative amounts can involve using measured signal to compare withmeasured signal from other samples. Absolute measurement can involvedetermining molar amounts or concentrations of nucleic acids in asample, for example, based on a standard curve.

Because label is introduced into nucleic acids in a stoichiometricmanner, relative amounts of amplified nucleic acids between samples canbe compared. If the detectable label is, for example, a fluorescentlabel, fluorescence from each sample can be measured. Absolute amountsof nucleic acid in a sample can be determined, for example, through theuse of a standard curve based on control samples. Alternatively,relative amounts of nucleic acids between samples can be determinedbased on relative quantity of detectable signal measured in each sample.

Colorimetry: Chemical reactions can generate products that producespecific color. At the molecular level, the absorption spectrum of theproduct is distinctly different from that of the substrate(s). Forexample, if the detectable label is an enzyme that generates a productwith absorption at a different wavelength than any of the reactants,then the detectable label can be indirectly quantified by the amountcolorimetric product produced.

In case primers with fluorescent labels are used to generate cDNA or DNAlibraries, the finished libraries will carry the label. In parallel,calibration standards could be prepared using the same method andreagents as the samples. The standards could be prepared in largeamounts and their molar concentration determined using anotherquantification method (e.g. UV absorbance or intercalating dyes) andknown molecular weight (MW). The MW can be computed if the standardshave a known length, or can be measured using an appropriate analyticalmethod, such as capillary electrophoresis (e.g. Fragment Analyzerinstrument). The standards could then be used to quantify libraries fromsamples using their fluorescent labels.

In certain embodiments, quantitation does not comprise providing anintercalating dye to nucleic acids and measuring an amount of dyeintercalated. In certain embodiments, quantitation does not compriseproviding labeled probes that hybridize to nucleic acid molecules andmeasuring an amount of probe hybridized to the nucleic acids.

E. Production of Libraries with Adjusted/Normalized Amounts ofPolynucleotides

Using these measurements, concentration of nucleic acids in a sample canbe adjusted to a predetermined or normalized amount. A plurality oflibraries can have normalized amounts or concentrations of nucleic acid.Normalized libraries can be used in pooled, normalized libraries.

In one embodiment, individual normalized libraries are produced by, forexample, diluting different samples to produce the desired molarity foreach sample. In some cases, the molarity will be the same for allsamples. Then, the same volumes of each normalized sample can becombined into a pool.

In another embodiment, volumes of different samples, each volumecontaining the same amount of nucleic acid, are removed from each sampleand combined into same volume second samples.

In another embodiment, volumes of different samples, each volumecontaining the same amount of nucleic acid, are combined into a commonpool to produce a pool of normalized libraries.

In subsequent steps as described herein, nucleic acid libraries in whichamounts of nucleic acid have been determined, or pooled libraries cannow be sequenced. Sequencing can proceed, for example, by highthroughput sequencing.

F. Sequencing Pooled Libraries

Sequencing nucleic acid libraries produces sequence reads of thepolynucleotides sequenced. Because nucleic acids in each nucleic acidlibrary bear a sample barcode sequence reads can be sorted into binsbased on the original library from which they are sourced. Sequencereads from individual libraries can be subject to further analysis. Inone embodiment, redundant sequences can be collapsed into an originalsequence, e.g., a nucleotide by nucleotide. Raw sequence reads orcollapsed reads may be referred to herein as “sequenced nucleic acids”.Sequenced nucleic acids in any library can be analyzed to determinequantities of target sequences in the sample. For example, if thelibrary comprises sequences of a microbiome, sequenced nucleic acids canbe analyzed to determine species present in the sample and amount ofeach species.

There are many bioinformatics methods that convert raw sequences intosecondary data. For example: Taxonomy classification uses databases withunique sequences belonging to different organisms. Once a sequence ismatched to the database, the presence of a specific organism can bedetected. By counting the sequences used to identify each organism,their relative abundances can also be measured. Functional assignmentscan also be made from the sequence reads. A database that correlatessequences to functions is used to convert sequencing reads intobiochemical functions.

G. Kits

Also provided herein are kits. Kits can comprise adapters and/or primersas disclosed herein. Kits can comprise reagents for performingbiochemical reactions, such as reagents for primer extension and/or foramplification, such as PCR. Such reagents can include any ofpolymerases, reverse transcriptases, nucleotides and buffers. Kits cancomprise containers for containing compositions of matter. Kits cancomprise containers comprising the aforementioned containers. Kits alsocan comprise instructions for use, e.g., printed instructions.

In some embodiments, kits comprise primers as described herein, in whicha portion of the primer comprise a detectable label and a portion do notcomprise a detectable label. The primers having a label can have thesame sequences as those not having a label. In some embodiments, the kitcan comprise first and second sets of primers/adapters. A first set canhave sequences that binding to a target primer binding site on a targetpolynucleotide. A second set can comprise sequences that bind to primerbinding sites on primers in the first set. In certain embodiments, onlyprimers in the second set bear detectable labels.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Preparation of Libraries from RNA

A method of preparing an RNA library comprises:

-   -   a) providing a sample containing: (i) from about 5 μL to about        100 μL (e.g., about 20 μL to about 50 μL) and (ii) an RNA        preservative [inhibits degradation of RNA];    -   b) optionally, disrupting cells in the sample;    -   c) isolating polynucleotides from the sample (e.g., on a silica        surface) (optionally, at this stage, non-informative RNA, such        rRNA and common host mRNA can be removed from the sample);    -   d) degrading DNA in the isolated polynucleotides (e.g., with a        DNase) to produce an RNA-enriched sample;    -   e) converting RNA into cDNA that contains primer sites for        forward and reverse primers    -   f) amplifying the cDNA of step e) using fixed molar amounts of        forward and reverse primers, where the amounts are the same,        through a sufficient number of amplification cycles to consume        all of the first and second primer.

Example 2 Normalized Nucleic Acid Libraries by Primer-LimitedAmplification

A plurality of different samples comprising RNA are provided; eachsample is in a separate container. To obtain a pooled normalized libraryof amplified nucleic acids from the samples

1. Each of the plurality of samples is treated as in Example 1, wherethe fixed molar amount of primer used in each sample is the same, or isrelated in a known ratio. Each sample is treated to label the nucleicacid with a barcode, where the barcodes for each sample are different.

2. A portion of each sample is removed and pooled with portions fromother samples. The relative molar amounts of nucleic acids from eachsample in the pooled sample is determined by the fixed molar amount ofprimer used to amplify each sample, and by the relative portion of eachsample added to the pooled sample.

Example 3 Normalized Nucleic Acid Libraries with Sequencing

1125 samples containing nucleic acid (e.g., RNA) were provided andamplified according to the primer-limited methods described herein, thensequenced on an Illumina sequencer. The mean number of reads was8,727,908. 1051 samples had a number of reads within 50% of the mean,representing 93.4% of the samples within the 50% cutoff. See FIG. 9.This represents far greater consistency than the current state of theart; see, e.g., Hosomichi et al., BMC Genomics 2014 15:645 (world wideweb site biomedcentral.com/1471-2164/15/645), in which ˜50% of thesamples were found to be within 50% of the mean.

Example 4 Normalized Libraries by Labeled Amplification

A sample comprising RNA is provided. To the sample is added a forwardprimer. The forward primer comprises a 3′ backspace.

1. During library preparation amplification step, add fluorescentprimers that amplify the cDNA fragments and incorporate fluorescence inthem:

Option A: Use only fluorescently-labeled PCR primers that containbarcodes.

Option B: Combine non-fluorescent primers with barcodes and shorterfluorescent primers that do not contain the barcode. This way, thefluorescent primers are universal for all libraries. Fraction of totalprimers that are fluorescent can be anywhere between 0-100%, dependingon the fluorescence yield of the fluorophore and the sensitivity of thefluorometer used to quantify the libraries.

2. Purify the libraries to remove any unincorporated fluorescentprimers. This can be done with commercially available kits.

3. Quantify the molar concentration of each library using a fluorometer.Use quantity standards for this purpose. Molarity can be computedbecause the fluorophores are added in a fixed stoichiometric ratio toall libraries. Average library length does not affect the fluorescencesignal.

4. (Optional) Cleave off the fluorescent tag if a cleavable linker wasused.

5. Normalize the libraries as needed based on the molar concentrationdetermined in step 3.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

While certain embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of amplifying nucleic acids comprisinga) performing a plurality of primer extension reactions on nucleic acidmolecules in each of a plurality of separate nucleic acid samplescontaining nucleic acids, using first polynucleotide primers to initiateprimer extension in each sample, wherein: 1) each of the plurality ofsamples is provided with a fixed molar amount of first primer; 2) thefixed molar amount of first primer in each sample is the same orsubstantially the same, or is related to the molar amount in othersamples in a known ratio; and 3) the plurality of primer extensionreactions in each sample is such that, upon completion of the pluralityof primer extension reactions, all first primer in each sample has beenused to produce amplified nucleic acids.
 2. The method of claim 1further comprising using a second polynucleotide primer, wherein thesecond primer is present in equal or substantially equal molar amount asthe first primer, or in a known greater molar amount as the firstprimer.
 3. The method of claim 2 wherein the second primer is present inequal or substantially equal molar amount as the first primer, and afterthe plurality of primer extension reactions the molar amount ofamplified nucleic acid is equal to or substantially equal to the molaramount of first and/or second primer.
 4. The method of claim 2 whereinthe first and second primers are different.
 5. The method of claim 1further comprising b) combining a portion of each of the plurality ofseparate samples to provide a pooled sample comprising a library ofamplified nucleic acids, wherein the molar amount of amplified nucleicacid from each separate sample in the pooled sample is the same orsubstantially the same, or related to the molar amounts of other nucleicacids in the pooled sample in a known ratio, thus producing a poolednucleic acid library.
 6. The method of claim 1 wherein the molar amountof first primer in each sample is the same or substantially the same. 7.The method of claim 1 wherein the nucleic acid molecules in theplurality of separate samples comprise RNA molecules.
 8. The method ofclaim 1 wherein the amplified nucleic acids comprise a sample barcodecomprising a predetermined nucleotide sequence, wherein the barcode isdifferent for each different nucleic acid sample.
 9. A method foramplifying nucleic acids comprising: a) providing a plurality of samplescomprising RNA; b) performing a first round of cDNA synthesis on the RNAin the samples to produce a library of first strand cDNA molecules; c)performing a second round of cDNA synthesis on the cDNA strand producedin the first round to provide second strand cDNA molecules, wherein thesecond strand cDNA molecules comprise a first primer binding site for afirst primer and a template for a second primer binding site for asecond primer; and d) amplifying the cDNA produced in step c) byproviding each of the plurality of samples a molar amount of firstprimer and second primer, wherein the molar amount of the second primeris equal to or substantially equal to the molar amount of the firstprimer, or is greater than the molar amount of the first primer in aknown amount, and performing a plurality of primer extension reactions,wherein 1) the molar amount of first primer in each of the plurality ofsamples is the same or substantially the same, or is related to themolar amount in other samples in a known ratio; and 2) wherein the molaramount of first primer in each sample is such that, upon completion ofthe plurality of primer extension reactions, all first primer in eachsample has been used.
 10. The method of claim 9 wherein the first primercomprises a first polynucleotide sequence and the second primercomprises a second polynucleotide sequence, wherein the first and secondpolynucleotide sequences are the same, and wherein the molar amount offirst and second primer in each sample is the same.
 11. The method ofclaim 9 wherein at least one of the primers comprises sequencingplatform-specific adapter sequences.
 12. The method of claim 11 whereinthe sequencing platform specific sequences comprise one or more of asequencing primer hybridization site, a sample barcode and a clusterprimer binding site.
 13. The method of claim 9 further comprising: e)preparing a pooled nucleic acid library by combining a portion of eachof the plurality of samples comprising amplified nucleic acid.
 14. Themethod of claim 9 wherein the predetermined molar amount of first primerin each of the plurality of samples is the same or substantially thesame.
 15. A method comprising a) preparing a set of nucleic acidlibraries by amplifying nucleic acids in a plurality of separate samplescomprising nucleic acids so that the amplified nucleic acids are presentin each of the plurality of separate samples in the same orsubstantially the same molar amount, or are present in molar amounts inknown ratios to each other; and b) combining a portion of each of theseparate samples to produce a pool of nucleic acid libraries in whichthe molar amounts of amplified nucleic acids from each nucleic acidlibrary in the pool are the same or substantially the same, or arepresent in molar amounts in known ratios to each other.
 16. The methodof claim 15 further comprising: c) sequencing the nucleic acids in thepool of nucleic acid libraries.
 17. The method of claim 15 wherein theamplified nucleic acids in the separate samples comprises a barcode,wherein the barcode for any nucleic acid library is the same for nucleicacids in that library and different from the barcodes for other nucleicacid libraries.
 18. A set of a plurality of separate nucleic acidlibraries, wherein: a) each library comprises polynucleotides; and b)molar amounts of polynucleotide molecules from each nucleic acid libraryin the set are the same or substantially the same, or are related as aknown ratio to each other.
 19. The set of nucleic acid libraries ofclaim 18, wherein each separate nucleic acid library is in a separatecontainer.
 20. The set of nucleic acid libraries of claim 18 furtherwherein b) the polynucleotides comprise a sample barcode, andpolynucleotides in any library have the same sample barcode; and c)sample barcodes in different libraries are different.
 21. The set ofnucleic acid libraries of claim 18 wherein the molar amounts ofpolynucleotide molecules in each library are the same or substantiallythe same.